Gardnerella pickettii sp. nov. (formerly Gardnerella genomic species 3) and Gardnerella greenwoodii sp. nov. (formerly Gardnerella genomic species 8) isolated from female urinary microbiome.

During an ongoing female urinary microbiome research study, strains c17Ua_112T and c31Ua_26T isolated from urine samples of a patient diagnosed with overactive bladder and a healthy postmenopausal woman, respectively, could not be allocated to any Gardnerella species with valid names. In this work, we aimed to characterize these strains. The 16S rRNA gene sequences confirmed that these strains are members of the genus Gardnerella. Phylogenetic analysis based on cpn60 strongly supported two clades, one encompassing c17Ua_112T and nine other strains from the public database, and the other including c31Ua_26T and three other strains, which were distinct from currently recognized species of the genus Gardnerella. Likewise, the phylogenomic tree also showed that strains c17Ua_112T and c31Ua_26T formed independent and robust clusters. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between c17Ua_112T and c31Ua_26T were 79.27 and 27.4 %, respectively. Strain c17Ua_112T showed the highest ANI (94.8 %) and dDDH values (59.8 %) with Gardnerella piotii UGent 18.01T, and strain c31Ua_26T revealed highest ANI (84.2 %) and dDDH (29.1 %) values with Gardnerella swidsinskii GS 9838-1T. Based on the data presented here, the two strains c17Ua_112T and c31Ua_26T represent two novel species of the genus Gardnerella, for which the names Gardnerella pickettii (c17Ua_112T=DSM 113414T=CCP 71T) and Gardnerella greenwoodii (c31Ua_26T=DSM 113415T=CCP 72T) are proposed.

Citrobacter meridianamericanus sp. nov., isolated from a soil sample.

A Gram-stain-negative strain, designated BR102T, isolated from a soil sample in Brazil was characterized by a polyphasic approach. Comparative 16S rRNA gene sequences indicated that strain BR102T belonged to the genus Citrobacter. The recN- and whole-genome-based phylogeny, and multilocus sequence analysis based on concatenated partial fusA, leuS, pyrG and rpoB sequences strongly supported a clade encompassing strain BR102T and a strain from public database that was distinct from currently recognized species of the genus Citrobacter. Average nucleotide identity and digital DNA-DNA hybridization values between strain BR102T and the closest relative Citrobacter freundii ATCC 8090T were 91.8 and 48.8 %, respectively. The ability to metabolize different compounds further discriminated strain BR102T from other closely related species of the genus Citrobacter. The novel variants bla CMY-179 and qnrB97, which encoded a CMY-2-like ß-lactamase and a QnrB-type protein, respectively, were identified in strain BR102T. BR102T was resistant to ampicillin, amoxicillin/clavulanate and cefoxitin. The DNA G+C content of strain BR102T is 51.3 mol%. Based on these results, strain BR102T represents a novel species of the genus Citrobacter, for which the name Citrobacter meridianamericanus sp. nov. is proposed. The type strain is BR102T (=MUM 22.55T=IMI 507229T).

Expanding the Bacterial Diversity of the Female Urinary Microbiome: Description of Eight New Corynebacterium Species.

The genus Corynebacterium is frequently found in the female urinary microbiome (FUM). In-depth characterization of Corynebacterium at the species level has been barely exploited. During ongoing FUM research studies, eight strains (c8Ua_144T, c8Ua_172T, c8Ua_174T, c8Ua_181T, c9Ua_112T, c19Ua_109T, c19Ua_121T, and c21Ua_68T) isolated from urine samples of healthy women or diagnosed with overactive bladder could not be allocated to any valid Corynebacterium species. In this work, we aimed to characterize these strains based on a polyphasic approach. The strains were Gram stain positive, rod to coccoid shaped, nonmotile, catalase positive, and oxidase negative. Phylogenetic analysis based on 16S rRNA and rpoB gene sequences indicated that all strains belonged to the genus Corynebacterium. The average nucleotide identity and digital DNA-DNA hybridization values among the genomes of the above eight strains and closely related type strains of the Corynebacterium genus were <95 (74.1%-93.9%) and <70% (22.2%-56.5%), respectively. Mycolic acids were identified in all strains. MK-8(H2) and/or MK-9(H2) were identified as the major menaquinones. The polar lipids' pattern mostly consisted of diphosphatidylglycerol, phosphatidylglycerol, and glycophospholipids. The major fatty acid was C18:1ω9c. Corynebacterium lehmanniae (c8Ua_144T = DSM 113405T = CCP 74T), Corynebacterium meitnerae (c8Ua_172T = DSM 113406T = CCP 75T), Corynebacterium evansiae (c8Ua_174T = DSM 113407T = CCP 76T), Corynebacterium curieae (c8Ua_181T = DSM 113408T = CCP 77T), Corynebacterium macclintockiae (c9Ua_112T = DSM 113409T = CCP 78T), Corynebacterium hesseae (c19Ua_109T = DSM 113410T= CCP 79T), Corynebacterium marquesiae (c19Ua_121T = DSM 113411T = CCP 80T), and Corynebacterium yonathiae (c21Ua_68T = DSM 113412T = CCP 81T) are proposed. This study evidenced that commonly used methodologies on FUM research presented limited resolution for discriminating Corynebacterium at the species level. Future research studying the biological mechanisms of the new Corynebacterium species here described may shed light on their possible beneficial role for healthy FUM.

Urinary Microbiome of Reproductive-Age Asymptomatic European Women.

The knowledge of bacterial species diversity within the female urinary microbiome (FUM) is essential for understanding the role of the FUM in urinary tract health and disease. This study aimed to characterize the bacterial species diversity of the FUM of asymptomatic reproductive-age European women by combining extended culturomics and long-read sequencing of the near-full-length 16S rRNA gene. A total of 297 bacterial species (median of 53 species/sample) were identified, yet only 22% of the species were detected by both culture and sequencing methods. Recently recognized Gardnerella, Lactobacillus, and Limosilactobacillus species and 5 new putative Corynebacterium species were identified by culturomics, while anaerobic species (e.g., 11 Peptoniphilus spp.) were mostly detected by amplicon sequencing. Notably, there was not a single species common to all samples, although members of the genus Lactobacillus were detected in all. Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus mulieris were observed in high relative abundance in several samples, as well as other species (e.g., Streptococcus agalactiae, Fannyhessea vaginae, Gardnerella vaginalis, Gardnerella swidsinskii), while low-abundance members (e.g., Finegoldia magna) were often more prevalent. A moderate correlation (Mantel test; r = 0.5) between community structure types captured by culturomics and amplicon sequencing was observed, highlighting the benefit of combining both methodologies. This study provided a detailed FUM structure at the species level, which is critical to unveil the potential relationship between specific microbiome members and urinary diseases/disorders. Moreover, the different capacity to characterize microbiome profiles of culturomic and amplicon sequencing is described, providing valuable insights for further urinary microbiome studies. IMPORTANCE The bacterial species diversity within the female urinary microbiome (FUM) has been insufficiently characterized. This study demonstrated that complementarity between optimized culture-dependent and -independent approaches is highly beneficial for comprehensive FUM species profiling by detecting higher FUM species diversity than previously reported, including identification of unreported species belonging to the genera Lactobacillus, Limosilactobacillus, and Latilactobacillus and putative novel Corynebacterium species. Although some species were present in high relative abundance, low-abundance members were more prevalent. FUM classification into community structure types demonstrated high interindividual differences in urinary microbiome composition among asymptomatic women. We also report moderate correlation between culture-dependent and -independent derived data-highlighting drawbacks of each methodological approach. Our findings suggest that FUM bacterial diversity reported from previous studies may be underestimated. Finally, our results contribute to the fundamental knowledge of the FUM required for further exploration of the urinary microbiome role in urinary tract diseases.

Citrobacter telavivum sp. nov. with chromosomal mcr-9 from hospitalized patients.

Strains 6105T and 6106, recovered from colonized patients in a hospital in Tel-Aviv, Israel, were compared with currently known species of the genus Citrobacter by a polyphasic taxonomic approach. Strains were characterized by whole-genome sequencing, 16S rRNA and recN gene sequencing, multilocus sequence analysis (MLSA), average nucleotide identity (ANI), Genome-to-Genome Distance Calculator (GGDC), and biochemical tests. The location and genetic surrounding of antibiotic resistance genes were investigated, and antibiotic susceptibility profiles were determined by broth microdilution or agar dilution methods. Phylogenetic analysis based on recN and MLSA revealed that both strains formed a distinct cluster from all currently recognized species. The ANI and GGDC were 90.7% and 54.3% with Citrobacter farmeri, respectively. The ability to metabolize various compounds also differentiated both strains from closely related Citrobacter species. Chromosomes of the isolates contained locus encoding a novel class A ß-lactamase (TEL-1; 90.5% amino acid identity with CdiA of Citrobacter koseri) plus a LysR-like transcriptional regulator (TEL-R) and an ~ 25.5-kb mcr-9 mosaic region. The direct mcr-9 context matched with those previously identified in several plasmids and chromosomes of diverse Enterobacteriaceae, yet similarity with the plasmidic loci extended further. Untypeable plasmids, pCTEL-2 (~ 235 kb) and pCTEL-1 (~ 114 kb), devoid of resistance genes, were identified in the strains. The isolates were non-susceptible to ß-lactams. The name Citrobacter telavivum sp. nov. is proposed, with 6105T (CECT 9989T or DSM 110286T) as the type strain. C. telavivum may represent a bacterial species adapting to hospital settings, able to disseminate and acquire antimicrobial resistance genes.

The status of the species Lactobacillus fornicalis Dicks et al. 2000. Request for an opinion.

During a recent study on members of the genus Lactobacillus we realized that cultures of Lactobacillus fornicalis TV 1018T (=DSM 13171T=ATCC 700934T) are no longer available from the online catalogue of the German Collection of Microorganisms and Cell Cultures GmbH, being displayed as Lactobacillus plantarum at the American Type Culture Collection. Based on data currently available, the organism deposited as ATCC 700934T is a member of the species Lactobacillus plantarum subs. plantarum. Therefore, the type strain of Lactobacillus fornicalis cannot be included in any further scientific comparative study. This matter is referred to the Judicial Commission, asking for an opinion on the status of the species.

Limosilactobacillus urinaemulieris sp. nov. and Limosilactobacillus portuensis sp. nov. isolated from urine of healthy women.

Two Gram-stain-positive strains, c9Ua_26_MT and c11Ua_112_MT, were isolated from voided urine samples from two healthy women. Comparative 16S rRNA gene sequences demonstrated that these novel strains were members of the genus Limosilactobacillus. Phylogenetic analysis based on pheS gene sequences and core genomes showed that each strain formed a separated branch and are closest to Limosilactobacillus vaginalis DSM 5837T. The average nucleotide identity (ANI) and Genome-to-Genome Distance Calculator (GGDC) values between c9Ua_26_MT and the closest relative DSM 5837T were 90.7 and 42.9 %, respectively. The ANI and GGDC values between c11Ua_112_MT and the closest relative DSM 5837T were 91.2 and 45.0 %, and those among the strains were 92.9% and 51,0 %, respectively. The major fatty acids were C12 : 0 (40.2 %), C16 : 0 (26.7 %) and C18 : 1 ω9c (17.7 %) for strain c9Ua_26_MT, and C18 : 1 ω9c (38.0 %), C16 : 0 (33.3 %) and C12 : 0 (17.6 %) for strain c11Ua_112_MT. The genomic DNA G+C content of strains c9Ua_26_MT and c11Ua_112_MT was 39.9 and 39.7 mol%, respectively. On the basis of the data presented here, strains c9Ua_26_MT and c11Ua_112_MT represent two novel species of the genus Limosilactobacillus, for which the names Limosilactobacillus urinaemulieris sp. nov. (c9Ua_26_MT=CECT 30144T=LMG 31899T) and Limosilactobacillus portuensis sp. nov. (c11Ua_112_MT=CECT 30145T=LMG 31898T) are proposed.

Citrobacter portucalensis sp. nov., isolated from an aquatic sample.

A Gram-stain-negative strain, A60T, isolated from a water well sample in Portugal, was characterized phenotypically, genotypically and phylogenetically. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain A60T belonged to the genus Citrobacter, and recN gene phylogeny revealed one strongly supported clade encompassing strain A60T and 13 other strains from public databases, distinct from currently recognized species of the genus Citrobacter. Furthermore, multilocus sequence analysis (MLSA) based on concatenated partial fusA, leuS, pyrG and rpoB sequences confirmed the classification obtained with the recN sequence. In silico genomic comparisons, including average nucleotide identity (ANI) and the genome-to-genome distance calculator (GGDC), showed 94.6 % and 58.4 % identity to the closest relative Citrobacter freundii ATCC 8090T, respectively. The ability to metabolize different compounds further discriminated strain A60T from other species of the genus Citrobacter. The G+C content of strain A60T is 52.0 %. The results obtained support the description of a novel species within the genus Citrobacter, for which the name Citrobacter portucalensis sp. nov. is proposed, with the type strain A60T (=DSM 104542T=CECT 9236T).

Prevalence of Mycobacterium avium subsp. paratuberculosis and Escherichia coli in blood samples from patients with inflammatory bowel disease.

Mycobacterium avium subsp. paratuberculosis (MAP) and adherent-invasive Escherichia coli (AIEC) have been implicated as primary triggers in Crohn's disease (CD). In this study, we evaluated the prevalence of MAP and E. coli (EC) DNA in peripheral blood from 202 inflammatory bowel disease (IBD) patients at various disease periods and compared against 24 cirrhotic patients with ascites (CIR) (non-IBD controls) and 29 healthy controls (HC). MAP DNA was detected by IS900-specific nested PCR, EC DNA by malB-specific nested PCR and AIEC identity, in selected samples, by sequencing of fimH gene. CD patients with active disease showed the highest MAP DNA prevalence among IBD patients (68 %). Infliximab treatment resulted in decreased MAP detection. CIR patients had high individual and coinfection rates (75 % MAP, 88 % EC and 67 % MAP and EC), whilst HC controls had lower MAP prevalence (38 %) and EC was undetectable in this control group. EC DNA prevalence in IBD patients was highly associated with CD, and 80 % of EC from the selected samples of CD patients analyzed carried the fimH30 allele, with a mutation strongly associated with AIEC. Our results show that coinfection with MAP and AIEC is common and persistent in CD, although the high MAP and EC detection in CIR patients suggested that colonization is, at least, partially dependent on increased gut permeability. Nevertheless, facilitative mechanisms between a susceptible host and these two potential human pathogens may allow their implication in CD pathogenesis.